PMID:8473341
| Citation |
Brown, ED and Wood, JM (1993) Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor by proline. J. Biol. Chem. 268:8972-9 |
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| Abstract |
The PutA protein is both the put repressor and a membrane-bound enzyme with proline and delta 1-pyrroline-5-carboxylate dehydrogenase activities. The conditions required for association of purified PutA protein with membrane vesicles suggested that a redox switching mechanism might determine the proportion of PutA protein functioning as a dehydrogenase (Wood, J. M. (1987) Proc. Natl. Acad. Sci. USA 84, 373-377). The FAD cofactor was released from the PutA protein with 1 M KBr at neutral pH. The apoprotein retained delta 1-pyrroline-5-carboxylate dehydrogenase and DNA binding but not proline dehydrogenase activity. Reconstitution with FAD fully restored proline dehydrogenase activity. Proline at a concentration of 0.11 mM caused half-maximal bleaching of the FAD in PutA. Chymotryptic digestion of the PutA protein in the presence and absence of proline demonstrated that the persistence of a 119-kDa protein fragment was characteristic of the reduced protein. Identical digestion patterns were obtained from the apoprotein in the presence and absence of proline. The quantity of the 119-kDa fragment produced varied with proline concentration, yielding a midpoint of 0.056 mM proline. The fraction of PutA protein associated with membrane vesicles was also a function of proline concentration, yielding a titration midpoint of 0.10 mM proline. Membrane binding was thus coincident with both flavin reduction and a change in protein conformation. |
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| Keywords |
Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Cell Membrane/metabolism; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Flavin-Adenine Dinucleotide/metabolism; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Oxidation-Reduction; Proline/metabolism; Protein Conformation |
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